Specific Aim 1: Determine whether NAM treatment alters hypoxic cell signals in human monocyte-derived macrophages (HMDMs) by: Probing for HIF1 protein levels, hypoxia response element transcriptional activity, and global gene expression changes via microarray analysis. Confirming possible changes at the gene level with protein assays. Determining whether exposure to NAM alters HMDM function, by probing phagocytosis and antigen presentation ability. Current Status: Treatment of HMDMs with NAM leads to loss of HIF1 protein after exposure to LPS. Concomitant with this change, hypoxia response element transcriptional activity is also decreased. Through global gene expression analysis, we also show that the HIF1 gene is downmodulated in the presence of NAM. We have also shown that macrophage genes associated with antigen presentation, such as CD80 and CD40, and myeloid modulation of immune responses, such as PD-L1 and PD-L2, are also decreased following NAM treatment. These changes are also present at the protein level upon exposure of HMDMs to NAM. Finally, we have shown that macrophages phagocytic activity is decreased in the presence of NAM. Specific Aim 2: Determine whether NAM treatment alters myeloid cell metabolism by: Exploring the redox potential (NAD/NADH) of HMDMs upon exposure to NAM. Determining the preferred metabolic pathways (lactate production and glucose uptake) of HMDMs upon NAM treatment. Probing global metabolomic changes. Current Status: In order to determine the metabolic effects of NAM, we have assayed the redox potential of HMDMs in the presence of NAM. The ratio of NAD to NADH is increased upon treatment with NAD and further mechanistic studies suggest this change to be due to activation of the NAD salvage and not the de novo pathway. We are currently in the process of further assaying aerobic glycolysis, oxidative phosphorylation, and glutathione metabolism. Global metabolomic studies are also underway. Specific Aim 3: Design studies to unravel possible mechanisms of NAM modulation of immune status. Current Status: In order to understand the mechanism by which NAM modulates HIF1, we have identified pathways that might be affected by NAM. Specifically, PHD2 is increased upon stimulation with NAM, suggesting that this might be a mechanism for increased HIF1 targeting for degradation. PHD2-mediated HIF1 degradation is through the proteasome. In fact, we can show that proteasome inhibition abrogates NAMs ability to decrease HIF1 levels, suggesting that NAM is acting through the proteasome. We are currently testing this hypothesis by investigating various steps in proteasome targeting of HIF1, such as ubiquitin activation, conjugation, and ligation. We have in vitro data suggesting that NAM induces changes in the immune reactivity of macrophages by possibly dampening excessive inflammation. Metabolomic profiling of immune cells exposed to NAM and LPS vs LPS alone has also been performed. These data suggest changes in aerobic glycolysis and oxidative phosphorylation. More in depth analysis is currently underway. Furthermore, we plan on further investigating the role of dietary compounds on immune homeostasis and function in vivo in animal models. Overall, this study has relevance in the context of inflammatory disorders where excessive inflammation is thought to play a role in the pathogenesis of disease, such as sepsis, autoimmunity, or immune-related adverse events. Projected Publication: Nicotinamide Regulation of Metabolism. Colleen S. Curran, Edward J. Dougherty, Parizad Torabi-Parizi. Manuscript in preparation.